Method and compositions for detection and enumeration of genetic variations

ABSTRACT

Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently-labeled particles via flow cytometry. Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and employed for further experimentation. This approach can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.

This application is a national stage application of co-pending PCTapplication PCT/US2004/015587 filed Jun. 9, 2004, which was published inEnglish under PCT Article 21(2) on Feb. 3, 2005, which claims thebenefit of application Ser. No. 60/485,301, filed Jul. 5, 2003 and60/525,859, filed Dec. 1, 2003, the contents of both of which areexpressly incorporated herein.

The invention disclosed herein was made using funds from the NationalInstitutes of Health grants CA 43460, CA 57345, and CA62924. The UnitedStates government therefore retains certain rights in the invention.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

FIELD OF THE INVENTION

The invention relates to the field of genetic analysis. In particular,it relates to methods and compositions for analyzing variations inindividual genes or transcripts and separating variants.

BACKGROUND OF THE INVENTION

The study of DNA sequence variation is essential for many areas ofresearch. The study of germ-line variations is essential for assessingthe role of inheritance in normal and abnormal physiologic states (1).Other variations, developed somatically, are responsible for neoplasia(2). The identification of such mutations in urine, sputum, and stoolcan therefore be used for the detection of presymptomatic cancers (3-5).Similarly, the detection of somatic mutations in lymph nodes, blood, orbone marrow can provide data about the stage of disease, prognosis, andappropriateness of various therapies (5). Somatic mutations innon-neoplastic cells also occur and appear to accumulate as humans ageor are exposed to environmental hazards (6). Such mutations occur inonly a small fraction of the cells in a tissue, thereby complicatingtheir analysis.

Central to the investigation of many of these issues is the detectionand quantification of sequence variants within a population of DNAmolecules. The number of molecules in each such collection is finite andtherefore countable.

Consider, for example, a collection of red and green balls. Countingthese balls is simple in principle but subject to basic probabilitytheory. If there is only one red ball for every 500 green balls, then itis necessary to count several thousand balls to get an accurate estimateof the proportion of red balls. If it is difficult to count enough ballsto make a reliable estimate, one can elute the paint off all the ballsand measure the color of the resultant paint mix.

In analogous fashion, small numbers of DNA molecules that vary by subtlechanges (single base pair substitutions or small deletions orinsertions) can be directly counted by amplifying individual DNAmolecules (single molecule PCR) (7-12). Such digital techniques havebeen shown to be extremely useful for measuring variation in genes ortheir transcripts. But digital technologies have so far been limited tocounting tens to thousands of molecules, either in the wells ofmicrotiter plates, on microscope slides, or after electrophoresis ofindividual PCR products. Analog techniques, analogous to the elution ofpaint from the balls described above, are generally easier to implementand can assess millions of molecules simultaneously (13). However, theiraccuracy and sensitivity is limited by instrumental and experimentalnoise. There is a continuing need in the art for methods which areaccurate and sensitive for measuring variation in genes or theirtranscripts.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment of the invention a composition is provided. Thecomposition comprises a plurality of beads. Each of the plurality ofbeads comprises a plurality of bound polynucleotides. Thepolynucleotides in the composition are heterogeneous; however, on atleast 1% of said beads the plurality of bound polynucleotides ishomogeneous.

In a second embodiment of the invention a liquid composition isprovided. The liquid composition comprises a plurality of microemulsionsforming aqueous compartments. At least a portion of said aqueouscompartments comprise a bead, a polynucleotide template, andoligonucleotide primers for amplifying the template. At least a portionof the oligonucleotide primers is bound to the bead.

A third embodiment of the invention provides a method for analyzingnucleotide sequence variations. Microemulsions comprising one or morespecies of analyte DNA molecules are formed. The analyte DNA moleculesin the microemulsions are amplified in the presence of reagent beadswhich are bound to a plurality of molecules of a primer for amplifyingthe analyte DNA molecules. Product beads are formed that are bound to aplurality of copies of a single species of analyte DNA molecule. Theproduct beads are separated from analyte DNA molecules which are notbound to product beads. A sequence feature of the single species ofanalyte DNA molecule that is bound to the product beads is determined.

A fourth embodiment of the invention is a probe for use in hybridizationto a polynucleotide that is bound to a solid support. The probecomprises an oligonucleotide with a stem-loop structure. At one of the5′ or 3′ ends there is a photoluminescent dye. The oligonucleotide doesnot comprise a quenching agent at the opposite 5′ or 3′ end.

A fifth embodiment of the invention is a pair of molecular probes. Thefirst and second probes each comprise an oligonucleotide with astem-loop structure having a first photoluminescent dye at one of the 5′or 3′ ends, and not comprising a quenching agent at the opposite 5′ or3′ end. The first oligonucleotide hybridizes to a wild-type selectedgenetic sequence better than to a mutant selected genetic sequence andthe second oligonucleotide hybridizes to the mutant selected geneticsequence better than to the wild-type selected genetic sequence. Thefirst and the second photoluminescent dyes are distinct.

In a sixth embodiment of the invention a method is provided forisolating nucleotide sequence variants. Microemulsions comprising one ormore species of analyte DNA molecules are formed. Analyte DNA moleculesin the microemulsions are amplified in the presence of reagent beads.The reagent beads are bound to a plurality of molecules of a primer foramplifying the analyte DNA molecules. Product beads are formed which arebound to a plurality of copies of one species of analyte DNA molecule.The product beads are separated from analyte DNA molecules which are notbound to product beads. The product beads which are bound to a pluralityof copies of a first species of analyte DNA molecule are isolated fromproduct beads which are bound to a plurality of copies of a secondspecies of analyte DNA molecule.

These and other embodiments of the invention, which will be apparentfrom the entire description of the invention, provide the art with theability to quantify genetic variations at a scale and ease heretoforeunattainable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the BEAMing method. Step 1—Magneticbeads covalently coated with streptavidin are bound to biotinylatedoligonucleotides (“oligos”). Step 2—An aqueous mix containing all thenecessary components for PCR plus primer-bound beads and template DNAare stirred together with an oil/detergent mix to create microemulsions.The aqueous compartments (white circles in the gray oil layer) containan average of <1 template molecule and <1 bead. Red and green templatesrepresent two template molecules whose sequences differ by one or manynucleotides. Step 3—The microemulsions are temperature cycled as in aconventional PCR. If a DNA template and a bead are present together in asingle aqueous compartment, the bead bound oligonucleotides act asprimers for amplification. The straight red and green lines connected tothe beads represent extension products from the two different kinds oftemplates. Step 4—The emulsions are broken and the beads are purifiedwith a magnet. Step 5—After denaturation, the beads are incubated witholigonucleotides that can distinguish between the sequences of thedifferent kinds of templates. Fluorescently-labeled antibodies are thenused to label the bound hybridization probes. This renders the beadscontaining PCR product as red or green upon appropriate laserexcitation. Step 6—Flow cytometry is used to count the red and greenbeads.

FIG. 2 is a photograph of a typical microemulsion. Microemulsions weremade as described infra with the exception that the aqueous compartmentscontained cascade blue-labeled dCTP and the beads were pre-labeled withR-phycoerythrin (red) or Alexa 488 (green). One microliter ofmicroemulsion was deposited in 1 microliter of oil on a microscope slideprior to photography. Of the seven aqueous compartments visible in thispicture, two contain beads. Note the heterogeneous size of the aqueouscompartments (beads are 1.05 microns in diameter).

FIG. 3A to FIG. 3D show density plots of flow cytometric data obtainedfrom BEAMing. The locus queried in this experiment was MID42 and PCRproducts generated from genomic DNA were used as templates in themicroemulsions. (FIG. 3A) Forward scatter (FSC) and side scatter (SSC)of all beads show that ˜80% of the total beads are singlets, with mostof the remaining beads aggregated as doublets. The “noise” isinstrumental and is observed with blank samples containing no beads. Theinstrument output was gated so that only singlets were analyzed forfluorescence analysis. The patterns observed from an individualhomozygous for the L allele (FIG. 3C), homozygous for the S allele (FIG.3B), and heterozygous for L and S (FIG. 3D) are shown. The regionscontaining beads hybridizing to the L and S allele probes are labeledgreen and red, respectively. The region containing beads that did nothybridize to any probe is black and the region containing beads thathybridized to both probes is blue. The blue beads arose from aqueouscompartments in which both types of template molecules were present. Theproportion of singlet beads that hybridized to at least one of theprobes was 2.9%, 4.3%, and 20.3% in (FIG. 3B) to (FIG. 3D),respectively. The FSC and SSC plots in (FIG. 3A) represent the samebeads analyzed in (FIG. 3D).

FIG. 4A to FIG. 4D show density plots of BEAMing using genomic DNA orRT-PCR products as templates. The data in (FIG. 4A) and (FIG. 4B) weregenerated by including 10 and 1 ug of human genomic DNA, respectively,in the microemulsions, querying the MID42 locus. The data in (FIG. 4C)and (FIG. 4D) were generated using emulsions that contained ˜50picograms of PCR products synthesized from cDNA of lymphoblastoid cells,querying the calpain-10 locus. The green and red regions correspond tothe L and S alleles for MID42 and to the A and G alleles for calpain-10.The number of beads in the outlined regions containing red or greenbeads is shown in each case. The proportion of singlet beads thathybridized to at least one of the probes was 1.2%, 0.6%, 6.8% and 4.2%in (FIG. 4A) to (FIG. 4D), respectively. The outlined regions used forcounting in (FIG. 4A) and (FIG. 4B) were identical, as were those usedfor (FIG. 4C) and (FIG. 4D). Beads that did not hybridize to any probewere gated out and therefore not evident in the graphs, while the regioncontaining beads that hybridized to both probes is labeled blue.

FIG. 5A to FIG. 5C show detection and validation of variants present ina minor fraction of the DNA population. (FIG. 5A) Mixtures of PCRproducts containing 0% to 4% L alleles of MID42 were used for BEAMing.Flow cytometry such as that shown in FIG. 3 was used to determine thefraction of singlet beads that were red (y-axis). The proportion ofsinglet beads that hybridized to at least one of the probes varied from3.2% to 4.3%. (FIG. 5B and FIG. 5C) Beads were sorted with the FACSVantage SE instrument and individual red or green beads were used astemplates for conventional PCR employing the forward and reverse primerslisted in FIG. 8. Red beads generated only the S allele sequence (FIG.5B; SEQ ID NO: 1) while green beads generated only the L allele sequence(FIG. 5C; SEQ ID NO: 2).

FIG. 6A to 6B demonstrate the use of agar in the aqueous phase of themicroemulsions. Emulsion bubbles that were formed by including 1.5%agarose in the aqueous compartment are shown. FIG. 6A shows the bubblesthat have fluorescents in them. FIG. 6B shows a darkfield image of thebubbles with one of the bubbles containing a bead in it. After breakingthe emulsions, the droplets containing magnetic beads can be recoveredby centrifugation and size fractionated through filtration or flowsorting.

FIG. 7 shows denaturing electrophoresis of two FAM-labeledoligonucleotides, 50 and 20 bases in length, which had been hybridizedto a 100 bp product on beads. The beads were embedded in an acrylamidegel in an oval shaped configuration and an electric field was appliedThe labeled oligonucleotides migrated off the beads and migrated adistance proportional to their size.

FIG. 8 shows oligonucleotides used.

DETAILED DESCRIPTION OF THE INVENTION

The inventors describe herein a digital technology, called BEAMing, thathas the power to assess millions of molecules and can be generallyapplied to the study of genetic variation. The technology involvesconversion of single DNA molecules to single beads each containingthousands of copies of the sequence of the original DNA molecule. Thenumber of variant DNA molecules in the population can then be assessed,for example, by staining the beads with fluorescent probes and countingthem using flow cytometry. Beads representing specific variants can beoptionally recovered through flow sorting and used for subsequentconfirmation and experimentation.

Beads according to the present invention are also known as microspheresor microparticles. Particle sizes can vary between about 0.1 and 10microns in diameter. Typically beads are made of a polymeric material,such as polystyrene, although nonpolymeric materials such as silica canalso be used. Other materials which can be used include styrenecopolymers, methyl methacrylate, functionalized polystyrene, glass,silicon, and carboxylate. Optionally the particles aresuperparamagnetic, which facilitates their purification after being usedin reactions.

Beads can be modified by covalent or non-covalent interactions withother materials, either to alter gross surface properties, such ashydrophobicity or hydrophilicity, or to attach molecules that impartbinding specificity. Such molecules include without limitation,antibodies, ligands, members of a specific-binding protein pair,receptors, nucleic acids. Specific-binding protein pairs includeavidin-biotin, streptavidin-biotin, and Factor VII-Tissue Factor.

Beads, after being prepared according to the present invention asproduct beads, have more than one copy of the same nucleic acid moleculebound to them. Preferably each bead is bound to at least 10, 50, 100,500, or 1000 molecules of the same nucleic acid sequence. In somecircumstances some of the product beads are bound to more than one typeof nucleic acid molecule. These product beads are generally less usefulin the analysis of ratios of genetic sequences in a population ofgenetic sequences. Such product beads can be readily discriminated andso will not distort the analysis.

A population of product beads will often comprise two or more types ofnucleic acids. Such a population is heterogeneous with respect to thenucleic acids. Desirably, a substantial proportion of the product beadscomprise only one type of nucleic acid per bead. A substantialproportion can be for example, at least 1%, at least 5%, at least 10%,or at least 50%. A product bead with only one type of nucleic acid perbead is termed homogeneous. Homogeneous beads with only one type ofnucleic acid per bead include those with nucleic acids containing errorsdue to errors in polymerase chain reaction. A product bead with twotypes of nucleic acid per bead is termed heterogeneous. Although notwishing to be bound by any particular theory, heterogeneous productbeads are thought to result from aqueous compartments which have morethan two molecules of template of non-identical sequence. A populationof product beads can be heterogeneous as a population but containindividual product beads that are homogeneous

Individual product beads preferably comprise more than one copy oftemplate analyte molecule. Each bead may comprise at least 10, at least50, at least 100, at least 500, or at least 1000 copies of templateanalyte. If the bead is homogeneous, each of those copies will beidentical.

Populations of product beads can be maintained in a liquid suspension.Alternatively they can be sedimented and dried or frozen. The latteralternatives may be beneficial for storage stability.

Analysis of populations of product beads can be useful fordistinguishing between many kinds of genetic variants. Polynucleotidescan be distinguished which differ by as little as a single nucleotidepolymorphism (SNP), by the presence or absence of a mutation, by thepresence or absence of an insertion or deletion, by the presence orabsence of a non-single nucleotide polymorphism. Thus populations ofproduct beads may be heterogeneous with regard to these geneticvariations.

One very convenient way for distinguishing genetic variants, i.e.,determining a sequence feature of the analyte, is by differentiallylabeling the variants with fluorescent dyes. Such labeling can beaccomplished by hybridization of a fluorescently labeled oligonucleotideprobe to one species of polynucleotide. Alternatively, a fluorescentlylabeled antibody can be used to specifically attach to oneoligonucleotide probe that hybridizes to a particular genetic variant.Such antibody binding can be, for example, mediated by a protein orpolypeptide which is attached to an oligonucleotide hybridization probe.Of course, other means of labeling polynucleotides as are known in theart can be used without limitation. Another means of labeling differentpolynucleotide species is by primer extension. Primers can be extendedusing labeled deoxyribonucleotides, such as fluorescently labeleddeoxyribonucleotides.

Populations of product beads can be used as templates. Template analytemolecules on the product beads can be analyzed to assess DNA sequencevariations by hybridization, primer-extension methods, massspectroscopy, and other methods commonly used in the art. Templateanalyte molecules on product beads can be employed for solid phasesequencing. In one solid phase sequencing technique, product beads arearrayed by placing them on slides spotted with complementaryoligonucleotides. In another solid phase sequencing technique, productbeads are placed into individual wells. In still another solid phasesequencing technique product beads are incorporated into acrylamidematrices (with or without subsequent polony formation). Sequencingreactions can be performed with any solid phase sequencing method, suchas those using unlabeled nucleotide precursors (e.g., pyrosequencing, asdescribed in Ronaghi et al., Anal. Biochem. 267: 65-71, 1999) or labelednucleotides (e.g., photocleavable reagents described by Mitra et al.,Anal. Biochem. 320:55-65, 2003). Product beads can thus be used for andfacilitate multiple parallel sequencing. Product beads can also be usedin sequencing employing Type IIS restriction endonucleases. Productbeads can also be used to provide templates for conventionaldideoxynucleotide sequencing. To obtain useful data upon sequenceanalysis, a homogeneous template population is desirable. To provide ahomogenous template population, product beads can be diluted, separated,or otherwise isolated so that each sequencing reaction contains a singleproduct bead. Alternatively, product beads can be sorted to providepopulations of beads with a single species of template.

Oligonucleotide primers can be bound to beads by any means known in theart. They can be bound covalently or non-covalently. They can be boundvia an intermediary, such as via a protein-protein interaction, such asan antibody-antigen interaction or a biotin-avidin interaction. Otherspecific binding pairs as are known in the art can be used as well. Toachieve optimum amplification, primers bound to the bead may be longerthan necessary in a homogeneous, liquid phase reaction. Oligonucleotideprimers may be at least 12, at least 15, at least 18, at least 25, atleast 35, or at least 45 nucleotides in length. The length of theoligonucleotide primers which are bound to the beads need not beidentical to that of the primers that are in the liquid phase. Primerscan be used in any type of amplification reaction known in the art,including without limitation, polymerase chain reaction, isothermalamplification, rolling circle amplification, self-sustaining sequencereplication (3SR), nucleic acid sequence-based amplification (NASBA),transcription-mediated amplification (TMA), strand-displacementamplification (SDA), and ligase chain reaction (LCR).

Microemulsions are made by stirring or agitation of oil, aqueous phase,and detergent. The microemulsions form small aqueous compartments whichhave an average diameter of 0.5 to 50 microns. The compartments may befrom 1 to 10 microns, inclusive, from 11 to 100 microns, inclusive, orabout 5 microns, on average. All such compartments need not comprise abead. Desirably, at least one in 10,000 of said aqueous compartmentscomprise a bead. Typically from 1/100 to 1/1 or from 1/50 to 1/1 of saidaqueous compartments comprise a bead. In order to maximize theproportion of beads which are homogeneous with respect tooligonucleotide, it is desirable that on average, each aqueouscompartment contains less than 1 template molecule. Aqueous compartmentswill also desirably contain whatever reagents and enzymes are necessaryto carry out amplification. For example, for polymerase chain reaction(PCR) the compartments will desirably contain a DNA polymerase anddeoxyribonucleotides. For rolling circle amplification a DNA polymeraseand a generic DNA circle may be present.

Emulsions can be “broken” or disrupted by any means known in the art.One particularly simple way to break the emulsions is to add moredetergent. Detergents which can be used include, but are not limited toTriton X100, Laureth 4, Nonidet.

Sample DNA for amplification and analysis according to the presentinvention can be genomic DNA, cDNA, PCR products of genomic DNA, or PCRproducts of cDNA, for example. Samples can be derived from a singleindividual, for example, from a body sample such as urine, blood,sputum, stool, tissue or saliva. Samples can also be derived from apopulation of individuals. The individuals can be humans, but can be anyorganism, plant or animal, eukaryotic or prokaryotic, viral ornon-viral.

Any type of probe can be used for specific hybridization to theamplified polynucleotides which are bound to the beads. Fluorescentlylabeled probes are useful because their analysis can be automated andcan achieve high throughput. Fluorescence activated cell sorting (FACS)permits both the analysis and the isolation of different populations ofbeads. One type of fluorescently labeled probe that can be used is amodified molecular beacon probe. These probes have stem-loop structuresand an attached fluorescent moiety on the probe, typically on one end ofthe probe, sometimes attached through a linker. Unlike standardmolecular beacon probes, modified molecular beacon probes do not have aquenching moiety. The modified molecular beacon probe can have thefluorescent moiety attached on either end of the probe, 5′ or 3′. Onesuch probe will hybridize better to a wild-type sequence than to amutant. Another such probe will hybridize better to a mutant sequencethan to the wild type. Still other probes will preferably hybridize toone polymorphic variant over another.

The method of the present invention provides a reliable and sensitiveassay for measuring variations in genes and transcripts. It requires noinstrumentation other than machines that are widely available. There areseveral other advantages of this approach. First, the sensitivity can beincreased to meet the particular specifications of an assay simply byanalyzing more beads. Such sensitivity is limited only by the error rateof the polymerases used for amplification. Second, the data obtained canbe used not only to demonstrate that a variant is present in aparticular population of DNA molecules, but also quantifies the fractionof variant DNA molecules in that population (FIG. 5A). Suchquantification is not possible with techniques that destroy or ignorethe wild type molecules as part of the assay, such as those that useallele specific priming or endonuclease digestion during PCR. Third, thebeads containing variant alleles can easily be purified through flowsorting. Such recovery is difficult with digital techniques that countmolecules deposited on microscope slides. And finally, the method isautomatable.

Several modifications of the basic principles described here can beenvisioned that will further simplify the technology or widen itsapplications. For example, microemulsions were made by stirringwater/oil/detergent mixes. The sizes of the resultant aqueouscompartments were somewhat heterogeneous, as illustrated in FIG. 2. Arelatively large number of beads containing PCR products of both allelesare obtained from large compartments because they are more likely tocontain >1 template molecule than smaller compartments. Though this isnot a problem for the analysis of uncommon variants, it does pose aproblem when the variant to be analyzed is present in a substantialfraction of the DNA molecules. For example, it is easy to distinguish apopulation containing 2% of allele A and 98% of allele B from one thatcontains 0% of allele A (FIG. 5A). But it is more difficult todistinguish a population that contains 48% of allele A and 52% of alleleB from a population that contains 50% of allele A; the large number ofheterozygote beads formed in the latter analysis diffuse the boundariesof the pure red and green channels. This limit to accuracy can beovercome through the preparation of more uniformly sized aqueouscompartments. Sonication or pressure-driven emulsifiers can make moreuniform compartments.

Though flow cytometry requires only seconds to minutes per sample,multiple parallel analyses could facilitate throughput. Novel particlecounting designs may prove useful for this purpose. Another way toincrease throughput would be to physically separate the beads thatcontained PCR products prior to flow cytometry. This could beaccomplished with proteins such as antibodies or streptavidin that bindto modified nucleotides incorporated into the PCR product duringamplification.

The methods of the invention can be applied to genes or transcripts ofany organism or population of organisms. These include withoutlimitation: humans, rodents, ungulates, mammals, primates, cows, goats,pigs, rats, mice, yeast, poultry, fish, shellfish, digs, cats,zebrafish, worms, algae. It can also be used to quantify epigeneticalterations, such as methylation, if DNA is first treated with bisulfiteto convert methylated cytosine residues to thymidine. Beads generatedfrom random fragments of whole genomes (24), rather than from individualgenes as described above, could be used to identify gene segments thatbind to specific DNA-binding proteins (25). And if the product beads areused in compartmentalized in vitro transcription-translation reactions,variant proteins can be bound to beads containing the correspondingvariant DNA sequences (23). This could allow facile flow cytometricevaluation of rare mutations using antibodies that distinguished betweenwild type and mutant gene products (26).

While the invention has been described with respect to specific examplesincluding presently preferred modes of carrying out the invention, thoseskilled in the art will appreciate that there are numerous variationsand permutations of the above described systems and techniques that fallwithin the spirit and scope of the invention as set forth in theappended claims.

EXAMPLES Example 1 Materials and Methods

Step 1—Coupling oligonucleotides to beads. Superparamagnetic beads of1.05+/−0.1 um in diameter, covalently bound to streptavidin, werepurchased from Dynal Biotech, Inc. (650.01, Lake Success, N.Y.). Beadswere washed once with 1×PCR buffer (53286, Invitrogen, Carlsbad, Calif.)then suspended in Bind and Wash Buffer (BWB) (5 mM Tris-HCl, 0.5 mMEDTA, 1.0 M NaCl, pH 7.5). Beads were incubated in BWB for 30 min atroom temperature in the presence of 10 uM oligonucleotides (FIG. 8).These oligonucleotides were modified with a dual biotin group at the 5′end with the biotin groups separated by a six-carbon linker (IDT,Coralville, Iowa). After binding, the beads were washed 3 times with1×PCR buffer to thoroughly remove unbound oligonucleotides.

Step 2—Preparing microemulsions. Microemulsions for PCR were prepared byslight modifications of previously described methods (14) (15). The oilphase was composed of 4.5% Span 80 (S6760, Sigma, St. Louis, Mo.), 0.40%Tween 80 (Sigma S-8074), and 0.05% Triton X-100 (Sigma T-9284) inmineral oil (Sigma M-3516). The oil phase was freshly prepared each day.The aqueous phase consisted of 67 mM Tris-HCl (pH 8.8), 16.6 mM NH4SO4,6.7 mM MgCl2, 10 mM β-mercaptoethanol, 1 mM dATP, 1 mM dCTP, 1 mM dGTP,1 mM dTTP, 0.05 uM forward primer, 25 uM reverse primer, 45 unitsPlatinum Taq (Invitrogen 10966-034), various amounts of template DNA(see results), and ˜10⁸ oligonucleotide-coupled beads in a total volumeof 300 ul. The forward primer was an oligonucleotide whose sequence wasidentical to the 3′ 20-22 nt of that described in step 1 and was notmodified with biotin.

Water-in-oil microemulsions were prepared by drop wise addition of 200microliters of the aqueous phase to 400 microliters of the oil phasepreviously placed in a 2 ml round bottom cryogenic vial (430661,Corning, Corning, N.Y.). The drop wise addition was performed over ˜oneminute while the mixture was being stirred at 1400 RPM with a magneticmicrostir bar (58948-353, VWR, Plainfield, N.J.) on a VWR model 565Magnetic Stirrer. After the addition of the aqueous phase, the mixturecontinued to be stirred for a total time of 30 minutes. Two emulsionswere made at once by placing two tubes in a rack placed at the center ofthe magnetic stirrer.

Step 3—PCR cycling. The emulsions were aliquoted into five wells of a 96well PCR plate, each containing 100 ul. PCR was carried out under thefollowing cycling conditions: 94° C. for 2 minutes; 40 cycles of 94° C.for 15 seconds, 57° C. for 30 seconds, 70° C. for 30 seconds. The PCRproducts analyzed in this study ranged from 189 to 239 bp.

Step 4—Magnetic capture of beads. After PCR cycling, the microemulsionfrom five wells of a PCR plate were pooled and broken by the addition800 microliters of NX buffer (100 mM NaCl containing 1% Triton X-100, 10mM Tris-HCl, pH 7.5, 1 mM EDTA) in a 1.5 ml tube (Corning 430909). Aftervortexing for ˜20 sec. the beads were pelleted by centrifugation in amicrocentrifuge at 8000 rpm (5000 g) for 90 seconds. The top oil phaseand all but ˜300 microliters of the aqueous phase was removed from thetube and 600 microliters of NX buffer was added. After vortexing for 20sec. and centrifugation for 90 sec., the top oil phase and all but ˜300microliters of the aqueous phase was removed. The addition of 600microliters NX buffer, vortexing, and centrifugation was repeated oncemore and the top oil portion and all but ˜300 microliters of the aqueousphase was removed. The tube was then placed on a magnet (Dynal MPC-S)and the rest of the supernatant was carefully pipetted off. The beadswere washed an additional 3 times with 1×PCR buffer using magneticseparation rather than centrifugation and finally resuspended in 100microliters of 1×PCR buffer.

Step 5—Sequence differentiation. Two oligonucleotide probes were usedfor each reaction. One was 5′-labeled with 6-carboxyfluorescein (6-FAM)and was specific for one allele while the second was 5′-labeled withbiotin and was specific for the other allele. Probes were synthesized byIDT. The 30 microliters hybridization reactions contained 10 uM of eachprobe and 5-25 million beads in 1×PCR buffer. Reactions were performedin PCR plates on a thermal cycler by heating to 94° C. for 30 secondsthen cooling to 75° C. at a rate of 0.5° C. per second, cooling to 45°C. at 0.2° C. per second, and finally cooled to 30° C. at 1° C. persecond. All subsequent steps were performed at room temperature. Thereactions were transferred to a 96 well Costar plate (Corning 3797) andplaced on a 96 well magnet. Beads were collected magnetically byexposing them to the magnet for 2 minutes. The supernatant was removedand the beads washed 3 times with 1×PCR buffer by pipetting them andcollecting for two minutes. They were finally resuspended in 100microliters B-PCR buffer (1 mg/mL BSA in 1×PCR buffer). The beads werethen incubated for 10 minutes in a total volume of 100 microliters B-PCRbuffer containing 3 ug of Alexa-488 rabbit anti-fluorescein antibody(Molecular Probes A-11090, Eugene, Oreg.) and 3 ug of Nutravidin labeledwith R-phycoerythrin (Molecular Probes A-2660) in B-PCR buffer. Thebeads were washed three times and resuspended in B-PCR buffer asdescribed above. They were then incubated for ten minutes in a totalvolume of 100 microliters B-PCR buffer containing 6 ug of Alexa488-conjugated chicken anti-rabbit antibody (Molecular Probes A-21441)and 3 ug of biotinylated goat anti-avidin antibody (BA-0300, VectorLaboratories, Burlingame, Calif.). The beads were washed three times andresuspended in B-PCR buffer as described above. They were then incubatedfor ten minutes in a total volume of 100 microliters B-PCR buffercontaining 3 ug of an Alexa 488-conjugated goat anti-chicken antibody(Molecular Probes A-11039) and 3 micrograms of R-phycoerythrin-labeledstreptavidin (Molecular Probes S-866). This solution was then washed anadditional 3 times with 1×PCR buffer and resuspended in 20 microlitersof 1×PCR buffer.

Step 6—Flow Cytometry. The bead suspension was diluted to aconcentration of ˜106-107 beads per ml in 10 mM Tris-HCl, 1 mM EDTA(351-010-131, Quality Biological, Inc., Gaithersburg, Md.) and analyzedusing a LSR instrument (BD Biosciences, Franklin Lakes, N.J.). Theinstrument was set up for standard two-color analysis using an argonlaser and optical filters that distinguished between the two fluorescentdyes. No spectral deconvolution was required as the major beadpopulations were well-separated. In some cases, scanning was performedwith FACScan or FACSCalibur instruments (BD Biosciences), yieldingequivalent results. Sorting was carried out with a FACS Vantage SEinstrument (BD Biosciences).

Template preparation and sequence analyses. Human genomic DNA waspurified with DNeasy (69504, Qiagen, Valencia, Calif.). RNA was purifiedwith Quickprep (27-9255-01, Amersham Biosciences, Piscataway, N.J.).Reverse transcription of RNA was performed using Superscript II reversetranscriptase (Invitrogen 18064014) according to the manufacturer'sinstructions. PCR using genomic DNA or reverse transcripts as templateswas performed as described (7). PCR products to be used as templates forBEAMing or for sequencing were purified with QIAquick (Qiagen 28104).Sequencing reactions were performed using Big Dye v3.0 reagents (AppliedBiosystems, Foster City, Calif.) and analyzed by capillaryelectrophoresis (Spectrumedix 9600, State College, Pa.).

Example 2 Results

Step 1—Coupling oligonucleotides to beads. We used streptavidin-beadsbecause of the simplicity of coupling biotinylated oligonucleotides tothem. Oligonucleotides with just a single 5′ biotin group were found todissociate from the beads during temperature cycling, whileoligonucleotides labeled with dual biotin groups at their 5′ end(separated by a six-carbon linker) were stable to cycling. As determinedby fluoroscopic measurements of oligonucleotides doubly labeled with6-FAM and biotin, ˜105 oligonucleotide molecules were bound to eachbead. We found that short oligonucleotides (20 bases) did not work aswell for priming as longer ones (41 bp), perhaps because of sterichindrance at the bead surface. It is likely that amino-, sulfhydryl-, orcarboxyl-modified oligonucleotides covalently coupled to beads modifiedwith corresponding reactive groups could also function as bead-boundprimers for BEAMing.

Step 2—Preparing microemulsions. The size of the individual aqueouscompartments ranged from less than 1 micron to >10 microns in diameter(FIG. 2). We estimated that an emulsion comprising 200 microliters ofaqueous solution and 400 microliters of oil would contain ˜3×10⁹compartments with an average diameter of 5 microns. Approximately 10⁸beads were included in each emulsion, so that only one in ˜30compartments contained a bead. The optimal amount of template wasexperimentally determined to be ˜5×10⁸ molecules, so that one in ˜sixcompartments contained a template molecule.

Step 3—PCR cycling. PCR priming by oligonucleotides coupled to beads wasfound to be very inefficient compared to the priming by the sameoligonucleotides when free in solution. For this reason, a small amountof non-biotinylated forward primer identical in sequence to thebiotinylated oligonucleotide coupled to the beads was included in thereactions. This facilitated the first few rounds of amplification of thesingle template within each aqueous compartment. In the absence ofadditional primer, no detectable amplification on the beads wasgenerated. Conversely, if too much additional primer was included, noamplification on the beads occurred because of competition with theprimers in solution. An excess of the reverse primer was included in theaqueous compartment to maximize the probability that bead-boundoligonucleotides extended by polymerase would serve as templates forfurther amplification cycles.

Step 4—Magnetic capture of beads. There are several ways to breakwater-in-oil emulsions, including extraction with organics (14). Wefound that simply adding non-ionic detergents produced phase separationswithout any detectable modification of the beads or DNA molecules boundto them. By measuring the amount of DNA that could be released from thebeads following restriction endonuclease digestion, we estimatethat >10,000 extended PCR products were present, on average, per bead.

Step 5—Sequence differentiation. Most fluorescence-based methods fordistinguishing alleles in homogeneous or two-phase assays can be used toassess allelic variation captured on beads. These methods include singlenucleotide extension, allele specific priming, or hybridization. Wegenerally employed hybridization of fluorescein-conjugated orbiotin-conjugated oligonucleotides for discrimination. As shown in FIG.1 and FIG. 8, these oligonucleotides had a stem-loop structure, with themiddle of the loop containing the variant nucleotide(s). This design wasbased on studies of Molecular Beacons wherein a stem-loop structure wasshown to markedly improve allelic discrimination (16). Theoligonucleotides we used differed from Molecular Beacons in that therewas no need for a quenching group. Such quenching is required forhomogeneous assays when unhybridized oligonucleotides cannot be removedfrom the reactions prior to assay but is not necessary for solid phaseassays such as those employed with beads.

Step 6—Flow Cytometry. Optimum results in flow cytometry depend on highfluorescent signals on the beads. We generally enhanced the fluorescenceemanating from the hybridization probes with secondary reagents. Forexample, Alexa 488-labeled antibodies were used to enhance the signalsemanating from fluorescein-coupled oligonucleotide probes. Similarly,R-phycoerythrin-labeled streptavidin was used to generate a signal frombiotin-labeled oligonucleotide probes. Flow cytometers equipped with twoor three lasers and appropriate filters have the capacity to distinguishmulti-allelic loci and to perform multiplex analysis of several genessimultaneously. The newest generation of flow cytometers can alsoanalyze >70,000 events per second. In addition to the analytical powerof flow cytometry, FACS instruments can separate specific populations ofbeads for further analysis.

Example 3 Characteristics of Microemulsions

Pilot experiments demonstrated that simply stirring the water-oilmixtures described in Materials and Methods produced very stablemicroemulsions of a size compatible with that of the beads. In theexperiment shown in FIG. 2, the aqueous compartment contained a blue dyeand 1 micron magnetic beads that were labeled by binding to anoligonucleotide that was biotinylated at its 5′ end and labeled withfluorescein at its' 3′ end The appearance of emulsions immediately aftertheir formation is shown in FIG. 2. As expected, this appearance wasunchanged after temperature cycling during PCR (15). Most aqueouscompartments contained no beads, as expected from the figures providedin the previous section. Those compartments that did contain beadsgenerally contained only one, though a fraction contained more, asexpected from a Poisson distribution and non-uniform aqueous compartmentsizes. “Heterozygous” beads containing PCR products representing bothalleles are produced when two or more DNA template molecules arecontained within a single aqueous compartment. Such heterozygotes cancompromise the accuracy of the analyses under some circumstances (seeDiscussion).

Example 4 Detection of Homozygotes and Heterozygotes

FIG. 3 shows typical results obtained with human DNA samples. The MID42marker used in this experiment was chosen from a collection of diallelicshort insertion/deletion polymorphisms assembled by Weber and colleagues(17). These alleles are particularly simple to distinguish withhybridization probes because the two alleles at each locus differ by ˜4bases. The probe for the longer (L) allele was labeled with fluorescein(green) and the probe for the shorter (S) allele labeled withR-phycoerythrin (red).

FIG. 3A shows a plot of the side scatter vs. forward scatter of beadsfollowing BEAMing. In general, >75% of beads were dispersed as singleparticles, with the remainder aggregated in groups of two or more.Subsequent flow cytometric analysis was confined to the singlet beads,gated as outlined in FIG. 3A.

FIGS. 3B-D show density plots of gated beads generated with varioustemplates. In FIG. 3B, a template from an individual homozygous for theL allele was included in the emulsion. Two populations of beads wereapparent. 98% of the beads contained no PCR product (black) and theremaining 2% fluoresced in the FL1 channel (colored green in FIG. 3).FIG. 3C represents the analysis of an individual homozygous for the Sallele. Two populations of beads were again apparent, but this time thelabeled population fluoresced in the FL2 channel (colored red in FIG.3). FIG. 3D presents density plots from the analysis of an individualheterozygous at the MID42 locus. Four populations of beads are evident:the black region represents beads without any PCR product, the redregion represents beads containing PCR products from the L allele, thegreen region represents beads containing PCR products from the S allele,and the blue region represents beads containing PCR products from bothalleles. Beads containing PCR products from both alleles were derivedfrom aqueous compartments which contained more than one templatemolecule. The number of blue beads increased in a non-linear fashion asmore template molecules were added. At the extreme, when all aqueouscompartments are saturated, virtually all beads will register as blue.Operationally, we found that the bead populations were most distinctwhen the number of beads containing any PCR product was <10% of thetotal beads analyzed.

Example 5 PCR Products, Genomic DNA or cDNA as Templates

The results shown in FIG. 3 were generated using PCR products made fromhuman genomic DNA samples. As the ratio of the beads representing Lalleles to those representing S alleles was 1.0 in this experiment, itwas clear that the initial PCR did not preferentially amplify eitherallele. The use of PCR products rather than genomic DNA permitted largenumbers of alleles to be amplified from even small quantities ofstarting DNA. In general, 10 to 100 picograms of PCR products of size200 bp were found to be optimal for BEAMing, producing PCR-mediatedextension of primers on ˜1 to 10% labeled beads.

In some situations it might be useful to use genomic DNA rather than PCRproducts as templates for BEAMing. The data in FIGS. 4A and B show flowcytometric data from an experiment wherein 10 ug or 1 ug of humangenomic DNA was used as template for BEAMing at the MID42 locus.Patterns very similar to those shown in FIG. 3 were observed, thoughfewer beads were labeled than when PCR products were used as templates.

BEAMing could also be used to analyze variations in expression from thetwo alleles of a heterozygous individual. Heritable variations in theexpression from individual alleles of the same gene have been shown tooccur often in humans (18) and mice (19) and can have significantphenotypic effects (20). The results shown in FIGS. 4C and D show thatPCR products made from reverse-transcribed mRNA can be used for BEAMing.In this case, calpain-10 transcripts differing by a single nucleotidepolymorphism (SNP) were analyzed. For SNPs like these, probes thatincorporated an extra mismatched nucleotide adjacent to the polymorphicnucleotide (see FIG. 8) can enhance the distinction between alleles (21)(22). The results from two independent emulsions made with aliquots ofthe same RT-PCR product are shown to illustrate reproducibility. Thoughthe number of beads that functioned as templates in BEAMing varied up to3-fold among experiments with identical templates, the proportion ofbeads representing the two alleles was reproducible (775 A allele beadsto 690 G allele beads in FIG. 4C and 1380 A allele beads to 1227 Gallele beads in FIG. 4D) respectively).

Example 6 Analysis of Minor Variants in a DNA Population

The analysis of uncommon variations is ideally suited for analysis viaBEAMing because of the large number of molecules that can beindependently analyzed while retaining a high signal-to-noise ratio.FIG. 5A shows representative data from templates representing 1%, 2%,3%, and 4% of the L allele of MID42. The linearity of thesemeasurements, with a correlation coefficient of 0.99, demonstrates theutility of this approach for such applications. We also applied thisanalysis to the detection of KRAS and could easily observe 0.1% mutantswhen spiked into a population of wt molecules (data not shown).

The rare beads representing the mutant alleles could not only bequantified but could also be purified for subsequent analysis. As ademonstration, samples of the beads enumerated in FIG. 5A wereadditionally assessed using a flow cytometer equipped with sortingcapabilities. Beads were sorted and individual beads used as templatesfor conventional PCR using the same primers employed for BEAMing. Aseach bead contains thousands of bound template molecules, single beadswere expected to generate robust PCR products (23) and this wasexperimentally confirmed. These PCR products were then subjected tosequencing. As shown in FIGS. 5B and C, green and red beads generatedPCR products exclusively of the L and S types, respectively.

Example 7 Electrophoresis of Oligonucleotides Hyrbridized to Beads

A 100 bp product was amplified on beads as described in Example 1, steps1 through 4. Two FAM-labeled oligonucleotides (50 and 20 bases inlength) were annealed to the 100 bp product on the beads. The beads werethen embedded in an acrylamide gel (using conventional Tris-Borate-EDTAelectrophoresis buffer) in an oval shaped configuration. An electricfield (250 V) was applied under denaturing conditions for 3 minutes. Thelabeled oligonucleotides migrated off the beads and migrated a distancerelated to their sizes. See FIG. 7. There was little diffusion, asevidenced by the retention of the oval shape of the beads.

Example 8 Sequencing of Templates Immobilized to Beads

Sanger-type (dideoxynucleotide) sequencing is performed using astemplates oligonucleotides which have been amplified on beads, asdescribed in Example 1. Individual beads are subjected to primerextension conditions in the presence of dideoxynucleotide inhibitors.The beads are then subjected to electrophoresis under denaturingconditions to separate the dideoxynucleotide-terminated, primer extendedoligonucleotides on the basis of length. A sequence is compiled based onthe length of the primer extended oligonucleotides.

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1. A method for analyzing nucleotide sequences, comprising: formingmicroemulsions comprising more than one species of analyte DNAmolecules, such that a plurality of aqueous compartments comprise asingle species of analyte DNA molecule; amplifying analyte DNA moleculesin the microemulsions in the presence of reagent beads, wherein thereagent beads are bound to a plurality of molecules of a primer foramplifying the analyte DNA molecules, whereby product beads are formedwhich are bound to a plurality of copies of the single species ofanalyte DNA molecule; separating the product beads from analyte DNAmolecules which are not bound to product beads; determining a sequencefeature of the single species of analyte DNA molecule which is bound tothe product beads by flow cytometry.
 2. The method of claim 1 whereinthe analyte DNA molecules are in a sample selected from the groupconsisting of urine, blood, sputum, stool, tissue, and saliva, of aeukaryotic organism.
 3. A method for analyzing nucleotide sequences,comprising: forming microemulsions comprising more than one species ofanalyte DNA molecules, such that a plurality of aqueous compartmentscomprise a single species of analyte DNA molecule; amplifying analyteDNA molecules in the microemulsions in the presence of reagent beads,wherein the reagent beads are bound to a plurality of molecules of aprimer for amplifying the analyte DNA molecules, whereby product beadsare formed which are bound to a plurality of copies of the singlespecies of analyte DNA molecule; separating the product beads fromanalyte DNA molecules which are not bound to product beads; determininga sequence feature of the single species of analyte DNA molecule whichis bound to the product beads; isolating product beads which are boundto a plurality of copies of the single species of analyte DNA;amplifying the single species of analyte DNA molecule from the isolatedproduct beads.
 4. The method of claim 3 wherein the analyte DNAmolecules are in a sample selected from the group consisting of urine,blood, sputum, stool, tissue, and saliva, of a eukaryotic organism.
 5. Amethod for analyzing nucleotide sequences, comprising: formingmicroemulsions comprising more than one species of analyte DNAmolecules, such that a plurality of aqueous compartments comprise asingle species of analyte DNA molecule; amplifying analyte DNA moleculesin the microemulsions in the presence of reagent beads, wherein thereagent beads are bound to a plurality of molecules of a primer foramplifying the analyte DNA molecules, whereby product beads are formedwhich are bound to a plurality of copies of the single species ofanalyte DNA molecule; separating the product beads from analyte DNAmolecules which are not bound to product beads; determining a sequencefeature of the single species of analyte DNA molecule which is bound tothe product beads by hybridization to oligonucleotide probes which aredifferentially labeled.
 6. The method of claim 5 wherein the analyte DNAmolecules are in a sample selected from the group consisting of urine,blood, sputum, stool, tissue, and saliva, of a eukaryotic organism.
 7. Amethod for analyzing nucleotide sequences, comprising: formingmicroemulsions comprising more than one species of analyte DNAmolecules, such that a plurality of aqueous compartments comprise asingle species of analyte DNA molecule; amplifying analyte DNA moleculesin the microemulsions in the presence of reagent beads, wherein thereagent beads are bound to a plurality of molecules of a primer foramplifying the analyte DNA molecules, whereby product beads are formedwhich are bound to a plurality of copies of the single species ofanalyte DNA molecule; separating the product beads from analyte DNAmolecules which are not bound to product beads; determining using flowcytometry an amount of product beads comprising the single species ofanalyte DNA molecule as a fraction of product beads.
 8. The method ofclaim 7 wherein the analyte DNA molecules are in a sample selected fromthe group consisting of urine, blood, sputum, stool, tissue, and saliva,of a eukaryotic organism.
 9. A method for isolating nucleotidesequences, comprising: forming microemulsions comprising more than onespecies of analyte DNA molecules, such that a plurality of aqueouscompartments comprise a single species of analyte DNA molecule;amplifying analyte DNA molecules in the microemulsions in the presenceof reagent beads, wherein the reagent beads are bound to a plurality ofmolecules of a primer for amplifying the analyte DNA molecules, wherebyproduct beads are formed which are bound to a plurality of copies of thesingle species of analyte DNA molecule; separating the product beadsfrom analyte DNA molecules which are not bound to product beads;isolating using fluorescence activated cell sorting product beads whichare bound to a plurality of copies of the single species of analyte DNAmolecule from product beads which are bound to a plurality of copies ofa second species of analyte DNA molecule.
 10. The method of claim 9wherein the analyte DNA molecules are in a sample selected from thegroup consisting of urine, blood, sputum, stool, tissue, and saliva, ofa eukaryotic organism.
 11. A method for isolating nucleotide sequences,comprising: forming microemulsions comprising more than one species ofanalyte DNA molecules, such that a plurality of aqueous compartmentscomprise a single species of analyte DNA molecule; amplifying analyteDNA molecules in the microemulsions in the presence of reagent beads,wherein the reagent beads are bound to a plurality of molecules of aprimer for amplifying the analyte DNA molecules, whereby product beadsare formed which are bound to a plurality of copies of the singlespecies of analyte DNA molecule; separating the product beads fromanalyte DNA molecules which are not bound to product beads; isolatingproduct beads which are bound to a plurality of copies of the singlespecies of analyte DNA molecule from product beads which are bound to aplurality of copies of a second species of analyte DNA molecule;amplifying the second species of analyte DNA molecule from the isolatedproduct beads.
 12. The method of claim 11 wherein the analyte DNAmolecules are in a sample selected from the group consisting of urine,blood, sputum, stool, tissue, and saliva, of a eukaryotic organism. 13.A method for analyzing nucleotide sequences, comprising: formingmicroemulsions comprising more than one species of analyte DNAmolecules, such that a plurality of aqueous compartments comprise asingle species of analyte DNA molecule; amplifying analyte DNA moleculesin the microemulsions in the presence of reagent beads, wherein thereagent beads are bound to a plurality of molecules of a primer foramplifying the analyte DNA molecules, whereby product beads are formedwhich are bound to a plurality of copies of the single species ofanalyte DNA molecule; separating the product beads from analyte DNAmolecules which are not bound to product beads; determining a sequencefeature of the single species of analyte DNA molecule which is bound tothe product beads by a technique selected from the group consisting of:hybridization to a fluorescently labeled oligonucleotide probe; allelespecific priming; single nucleotide extension; hybridization to afluorescein-conjugated oligonucleotide probe; and hybridization to abiotin-conjugated oligonucleotide probe.
 14. The method of claim 13wherein the analyte DNA molecules are in a sample selected from thegroup consisting of urine, blood, sputum, stool, tissue, and saliva, ofa eukaryotic organism.
 15. The method of claim 13 wherein the techniqueused for determining is hybridization to a fluorescently labeledoligonucleotide probe.
 16. The method of claim 15 wherein theoligonucleotide probe has a stem and loop structure.
 17. The method ofclaim 13 wherein the technique used for determining is allele specificpriming.
 18. The method of claim 13 wherein the technique used fordetermining is single nucleotide extension.
 19. The method of claim 13wherein the technique used for determining is hybridization to afluorescein-conjugated oligonucleotide probe.
 20. The method of claim 19wherein the oligonucleotide probe has a stem and loop structure.
 21. Themethod of claim 13 wherein the technique used for determining ishybridization to a biotin-conjugated oligonucleotide probe.